1 Review 2 3 Diverse midbrain dopaminergic neuron subtypes and implications for complex 4 clinical symptoms of Parkinson's disease 5 6 Kathleen Carmichael1,2, Breanna Sullivan1, Elena Lopez1, Lixin Sun1, Huaibin 7 Cai1, * 8 9 1Transgenic Section, Laboratory of Neurogenetics, National Institute on Ageing, 10 National Institutes of Health, Bethesda, MD 20892, USA. 11 2The Graduate Partnership Program of NIH and Brown University, National Institutes 12 of Health, Bethesda, MD 20892, USA. 13 14 Correspondence to: Dr. Huaibin Cai, Transgenics Section, Laboratory of 15 Neurogenetics, National Institute on Ageing, National Institutes of Health, Building 35, 16 Room 1A112, MSC 3707, 35 Convent Drive, Bethesda, MD 20892–3707, USA. E-mail: 17 [email protected] 18 19 How to cite this article: Carmichael K, Sullivan B, Lopez E, Sun L, Cai H. Diverse 20 midbrain dopaminergic neuron subtypes and implications for complex clinical 21 symptoms of Parkinson's disease. Ageing Neur Dis 2021;1:[Accept]. 22 http://dx.doi.org/10.20517/and.2021.07 23 24 Received: 16 Jun 2021 Revised: 12 Jul 2021 Accepted: 14 Jul 2021 First online: 25 15 Jul 2021 26 27 28 Abstract 29 Parkinson's disease (PD), the most common degenerative movement disorder, is 30 clinically manifested with various motor and non-motor symptoms. Degeneration of 31 midbrain substantia nigra pas compacta (SNc) dopaminergic neurons (DANs) is 32 generally attributed to the motor syndrome. The underlying neuronal mechanisms of 33 non-motor syndrome is largely unexplored. Besides SNc, midbrain ventral tegmental 34 area (VTA) DANs also produce and release dopamine and modulate movement, reward, 35 motivation, and memory. Degeneration of VTA DANs also occurs in postmortem brains 36 of PD patients, implying an involvement of VTA DANs in PD-associated non-motor 37 symptoms. However, it remains to be established that there is a distinct segregation of 38 different SNc and VTA DAN subtypes in regulating different motor and non-motor 39 functions, and that different DAN subpopulations are differentially affected by normal 40 ageing or PD. Traditionally, the distinction among different DAN subtypes was mainly 41 based on the location of cell bodies and axon terminals. With the recent advance of 42 single cell RNA sequencing technology, DANs can be readily classified based on 43 distinct gene expression profiles. A combination of distinct anatomic and molecular 44 markers shows great promise to facilitate the identification of DAN subpopulations 45 corresponding to different behavior modules under normal and disease conditions. In 46 this review, we will first summarize the recent progress in characterizing genetically, 47 anatomically, and functionally diverse midbrain DAN subtypes. Then we will provide 48 perspectives on how the preclinical research on the connectivity and functionality of 49 DAN subpopulations improves our current understanding of cell-type and circuit 50 specific mechanisms of the disease, which could be critically informative for designing 51 new mechanistic treatments. 52 53 Keywords: Parkinson's disease, ageing, dopaminergic neurons, dopamine, SNc, VTA, 54 ALDH1A1, RNA sequencing, subpopulation 55 56 57 INTRODUCTION 58 Parkinson’s disease (PD) is the second most common degenerative neurological 59 disorder affecting millions of elderly individuals worldwide. PD patients display 60 canonical motor symptoms, including resting tremor, slowed movement, impaired 61 posture and balance, and rigid muscles[1]. The motor syndrome is generally regarded as 62 the result of extensive loss of nigrostriatal dopaminergic neurons (DANs) in the 63 substantia nigra pars compacta (SNc) of the midbrain[2, 3]. Dopamine replacement 64 medications and deep brain stimulation surgery can improve some of patient’s motor 65 conditions. However, no cure is available. Moreover, long-term medication can cause 66 severe side effects, such as dyskinesia and impulsive control disorders [4]. New 67 mechanistic insights and therapeutic agents are still needed to improve patients’ 3 Surname et al. Ageing Neur Dis Year;Volume:Number│http://dx.doi.org/10.20517/and.xxx.xx Page X of 6 68 treatment and life quality. 69 70 In addition to motor symptoms, PD patients often suffer from depression, dementia, 71 and other neuropsychiatric symptoms [5]. For example, PD patients often develop 72 cognitive dysfunctions, which leads to Parkinson’s disease dementia (PDD) [6, 7]. 73 Approximately 75% of PD patients develop dementia within 10 years of diagnosis, and 74 the prevalence of PDD is 0.3-0.5% in the general population older than 65 years [6]. The 75 exact pathogenic mechanisms of PDD and other PD-related non-motor symptoms are 76 largely unknown. The cognitive dysfunctions are not improved by levodopa, the most 77 effective drug to treat the motor symptoms in PD [6]. Therefore, an important step in 78 intervening complex neurological disorder like PD, is to fully elucidate the functional 79 roles of different neural circuits responsible for specific behavioral phenotypes. 80 81 It has been generally accepted that ageing, environmental toxins and genetic mutations 82 contribute to the etiopathogenesis of PD. Ageing is the most significant risk factor in 83 the development of PD and other neurodegenerative diseases. Genomic instability, 84 epigenetic alterations, loss of proteostasis, mitochondrial dysfunction and altered 85 intracellular communication are among the key ageing hallmarks [8]. Various 86 environmental toxins and genetic risk factors have been linked to PD, which affect 87 largely overlapping molecular and cellular pathways as implicated in ageing [9-11]. 88 However, the molecular genetic studies often fall short in pinpointing any specific 89 cell-types or neural circuits critical for the alterations and impairments of motor and 90 non-motor behaviors. To better understand how different brain cells and neural circuits 91 control diverse behaviors will therefore provide the structural framework to better 92 appreciate the impact of ageing, environmental and genetic factors on the cause and 93 progression of the disease-related behavioral abnormalities. 94 95 The midbrain DANs are composed of diverse neuron subpopulations based on the 96 location of cell bodies, projection patterns, morphology, gene expression profiles, 97 electrophysiological properties, physiological functions, and vulnerabilities to diseases 98 [12-17]. Since a preferential degeneration of midbrain DANs represents the most 99 significant neuropathological feature of PD, in this review we focus our discussion 100 mainly on the diversity of midbrain DANs in their distinct genetic makeups, 101 connectivity and functionality. Because animal research has been instrumental in 4 102 understanding the pathophysiological mechanisms and developing the treatments of PD, 103 such as the current dopamine replacement therapy and deep brain stimulation 104 surgery[18], we will mainly summarize the research findings from preclinic animal 105 models. 106 107 MOLECULAR GENETIC DIVERSITY OF MIDBRAIN DOPAMINERGIC 108 NEURONS 109 Midbrain DANs, residing in the ventral region of the midbrain, are grouped together 110 based on their ability to synthesize and release dopamine, a key neuromodulator 111 involved in motor control and learning, motivation, cognition, and reward [19, 20]. 112 Dysfunction of midbrain DAN-mediated dopamine transmission has been associated 113 with PD, schizophrenia, addiction, and other neurological and psychological disorders 114 [5, 21, 22]. Traditionally, midbrain DANs can be divided into three main subgroups, 115 retrorubral field (RRF, A8), SNc (A9), and ventral tegmental area (VTA, A10), in 116 humans and rodents [23, 24]. The midbrain DANs also differ in their axon projections to 117 different brain regions and physiological functions. In addition, the SNc DANs are 118 relatively more vulnerable to neuronal toxins in rodent PD models and preferentially 119 degenerated in PD patients [25, 26]. However, molecular genetic makers are needed to 120 better characterize different DAN subtypes, and their distinctive connectivity and 121 functionality. 122 123 The midbrain DANs selectively express genes critical for the dopamine synthesis, 124 transport, and degradation, such as tyrosine hydrolase (TH), vesicular monoamine 125 transporter 2 (VMAT2), dopamine transporter (DAT), and aldehyde dehydrogenase 1a1 126 (ALDH1A1) [27]. The midbrain DANs also express transcription factors critical for the 127 DAN differentiation and survival, including nuclear receptor related 1 protein 128 (NURR1), pituitary homeobox3 (PITX3) and forkhead box protein A1/2 (FOXA1/2) [28, 129 29]. The molecular genetic difference between SNc and VTA DANs was initially studied 130 by immunostaining and in situ hybridization using preselected genetic makers. Those 131 markers included G-protein activated inwardly rectifying potassium channel 2 132 (GIRK2/KCNJ6), which is more abundant in SNc DANs [30, 31], and calbindin (D-28K, 133 CALB1), which is more enriched in VTA DANs [32-34]. However, those genetic markers 134 are not exclusively expressed by the SNc or VTA DANs. Additional genetic markers 135 are required to specifically investigate the functions of different SNc and VTA DAN 5 Surname et al. Ageing Neur Dis Year;Volume:Number│http://dx.doi.org/10.20517/and.xxx.xx Page X of 6 136 subtypes. The whole-genome gene expression studies using microarray and RNA 137 sequencing (RNAseq) technology, especially the latest single cell RNAseq (scRNAseq) 138 techniques,